Our recent findings made during this funding period have revealed an unexpected dependence of canonical Burkitt's Lymphomas (BLs) and Wp-restricted BLs on EBV's BART miRNAs (Vereide and Sugden, 2011;Vereide et al., submitted). We have also found that EBV's miRNAs promote transformation of newly infected B-cells (Seto et al., 2010;Vereide et al., submitted). We propose to identify the mRNAs that are regulated by EBV's miRNAs to drive tumor maintenance and promote transformation. EBV encodes 25 pre-miRNAs which can yield 50 mature miRNAs. These miRNAs have predicted seed sites in more than 30% of all human mRNAs. However, many studies, including our own, indicate that the vast majority of these mRNAs are not regulated by EBV's miRNAs (Kuzembayeva et al. submitted;Dolken et al. 2010). It is therefore essential to develop functional assays to allow characterization of presumptive targets of miRNAs in order to learn if the subtle differences in their expression mediated by miRNAs have functional consequences. We have developed functional assays for tumor maintenance and transformation dependent on EBV's miRNAs when the miRNAs are expressed at physiological levels. This latter qualification is important because miRNAs act in a dose-dependent manner, and EBV's miRNAs are often expressed at low levels (Pratt et al., 2009). We propose to identify minimal subsets of EBV's miRNAs that regulate a given phenotype and then develop matched pairs of cells that do and do not express these miRNAs from EBV plasmids. The mRNAs in the RISCs immunoprecipitated from these pairs of cells will be identified and enumerated by deep sequencing, sorted bioinformatically, and their 3'UTRs tested in reporter assays for regulation by specific viral miRNAs. We have used all of these assays successfully in our identification of caspase 3 as a target for BART miRNAs in canonical BLs (Vereide et al. submitted). The mRNAs found by this set of assays will then be tested functionally for potential contributions to maintaining lymphomas and transforming primary B-cells.

Public Health Relevance

Epstein - Barr virus (EBV) causes lymphomas and carcinomas in people throughout the world. No vaccine is available for preventing infection by EBV. We are identifying and characterizing those EBV genes that allow tumors to survive and proliferate to elucidate viral targets for developing anti-viral, anti-tumor therapies. These studis are also revealing how EBV, which is a paradigm for human tumor viruses, sustains tumors. EBV is exceptional for the number of miRNAs it encodes. We have now found that these small RNAs contribute both to tumor maintenance and to transformation of newly infected B-cells and propose to elucidate their mechanisms of driving this tumorigenesis and transformation.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Daschner, Phillip J
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University of Wisconsin Madison
Internal Medicine/Medicine
Schools of Medicine
United States
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Kuzembayeva, Malika; Hayes, Mitchell; Sugden, Bill (2014) Multiple functions are mediated by the miRNAs of Epstein-Barr virus. Curr Opin Virol 7:61-5
Shrestha, Prabha; Sugden, Bill (2014) Identification of properties of the Kaposi's sarcoma-associated herpesvirus latent origin of replication that are essential for the efficient establishment and maintenance of intact plasmids. J Virol 88:8490-503
Vereide, D T; Seto, E; Chiu, Y-F et al. (2014) Epstein-Barr virus maintains lymphomas via its miRNAs. Oncogene 33:1258-64
Woellmer, Anne; Hammerschmidt, Wolfgang (2013) Epstein-Barr virus and host cell methylation: regulation of latency, replication and virus reactivation. Curr Opin Virol 3:260-5
Chiu, Ya-Fang; Sugden, Arthur U; Sugden, Bill (2013) Epstein-Barr viral productive amplification reprograms nuclear architecture, DNA replication, and histone deposition. Cell Host Microbe 14:607-18
Pratt, Zachary L; Zhang, Jingzhu; Sugden, Bill (2012) The latent membrane protein 1 (LMP1) oncogene of Epstein-Barr virus can simultaneously induce and inhibit apoptosis in B cells. J Virol 86:4380-93
Vereide, David T; Sugden, Bill (2011) Lymphomas differ in their dependence on Epstein-Barr virus. Blood 117:1977-85
Seto, Eri; Moosmann, Andreas; Gromminger, Sebastian et al. (2010) Micro RNAs of Epstein-Barr virus promote cell cycle progression and prevent apoptosis of primary human B cells. PLoS Pathog 6:e1001063
Vereide, David; Sugden, Bill (2010) Insights into the evolution of lymphomas induced by Epstein-Barr virus. Adv Cancer Res 108:1-19
Bergbauer, Martin; Kalla, Markus; Schmeinck, Anne et al. (2010) CpG-methylation regulates a class of Epstein-Barr virus promoters. PLoS Pathog 6:e1001114

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